Transfusion of red blood cells (RBCs), the most common therapeutic procedure in US hospitals, is effective at preventing morbidity and mortality in anemic patients. However, stored RBC units progressively develop functional defects known as RBC storage lesions that may affect clinical outcomes. Donor-specific factors may also determine the transfusion efficacy of stored RBCs. Because transfusion of RBC units that are defective, due to storage time and/or donor factors, may adversely affect thousands of patients annually, approaches should be developed to screen these units from the blood supply. Metabolomics is a powerful approach to identify biomarkers that correlate with metabolic and functional changes in stored RBCs. In support of our objective to identify metabolic biomarkers for donor testing, we have now demonstrated that AS1-RBCs from > 90% of donors show typical changes in hundreds of metabolites during 42 day storage (the typical metabotype), whereas the remaining donors have markedly divergent metabotypes. Intriguingly, there is not single atypical metabotype: all donors we have tested to date with atypical metabotypes differed significantly from one another. Thus, we propose that these divergent metabotypes differentiate a small percentage of stored RBCs as metabolic/functional outliers, and that metabolic alterations identified in their RBCs can be used to develop biomarkers to detect RBCs with different survival or efficacy characteristics after transfusion. In the proposed studies, we will utilize RBCs from known-poor storing donors to identify metabolic biomarkers that correlate with several measures of in vitro dysfunction during storage. These biomarkers will then be used to screen random donors, some of whom will also be tested for in vivo RBC survival after transfusion to test whether the candidate biomarkers (which were initially identified from known poor-storing donors) can in turn identify randomly selected donors with poor-storage characteristics. Successful completion of this work will result in identification of a series of RBC biomarkers that predict altered RBC function during blood storage, that can be further validated for clinical use, and that can be used as a starting point to dissect mechanisms underlying poor-storage characteristics of donated RBCs.